Various methods of labelling molecules of interest are known in the art, including radioactive atoms, fluorescent dyes, luminescent reagents, electron capture reagents and light absorbing dyes. Each of these labelling systems has features which make it suitable for certain applications and not others. More recently there has been development in the area of mass spectrometry as a method of detecting labels that are cleavably attached to their associated molecule of interest.
For many applications such as nucleic acid analysis, the structure of the analyte can be determined from indirect labelling. This is advantageous particularly with respect to the use of mass spectrometry because complex biomolecules such as DNA have complex mass spectra and are detected with relatively poor sensitivity. Indirect detection means that an associated label molecule can be used to identify the original analyte, where the label is designed for sensitive detection and a simple mass spectrum. Simple mass spectra mean that multiple labels can be used to analyse multiple analytes simultaneously.
PCT/GB98/00127 describes arrays of nucleic acid probes covalently attached to cleavable labels that are detectable by mass spectrometry which identify the sequence of the covalently linked nucleic acid probe. The labelled probes of this application have the structure Nu-L-M where Nu is a nucleic acid covalently linked to L, a cleavable linker, covalently linked to M, a mass label. Preferred cleavable linkers in this application cleave within the ion source of the mass spectrometer. Preferred mass labels are substituted poly-aryl ethers. This application discloses a variety of ionisation methods and analysis by quadrupole mass analysers, Time of Flight (TOF) analysers and magnetic sector instruments as specific methods of analysing mass labels by mass spectrometry.
PCT/GB94/01675 discloses ligands, and specifically nucleic acids, cleavably linked to mass tag molecules. Preferred cleavable linkers are photo-cleavable. This application discloses Matrix Assisted Laser Desorption Ionisation (MALDI) Time of Flight (TOF) mass spectrometry as a specific method of analysing mass labels by mass spectrometry.
PCT/US97/22639 discloses releasable non-volatile mass-label molecules. In preferred embodiments these labels comprise polymers, typically biopolymers which are cleavably attached to a reactive group or ligand, i.e. a probe. Preferred cleavable linkers appear to be chemically or enzymatically cleavable. This application discloses MALDI TOF mass spectrometry as a specific method of analysing mass labels by mass spectrometry.
PCT/US97/01070, PCT/US97/01046, and PCT/US97/01304 disclose ligands, and specifically nucleic acids, cleavably linked to mass tag molecules. Preferred cleavable linkers appear to be chemically or photo-cleavable. These applications disclose a variety of ionisation methods and analysis by quadrupole mass analysers, TOF analysers and magnetic sector instruments as specific methods of analysing mass labels by mass spectrometry.
None or these prior art applications mention the use of tandem or serial mass analysis of tagged biomolecules.
Gygi et al. (Nature Biotechnology 17: 994-999, “Quantitative analysis of complex protein mixtures using isotope-coded affinity tags” 1999) disclose the use of ‘isotope encoded affinity tags’ for the capture of peptides from proteins, to allow protein expression analysis. In this article, the authors describe the use of a biotin linker, which is reactive to thiols, for the capture peptides with cysteine in them. A sample of protein from one source is reacted with the biotin linker and cleaved with an endopeptidase. The biotinylated cysteine-containing peptides can then be isolated on avidinated beads for subsequent analysis by mass spectrometry. Two samples can be compared quantitatively by labelling one sample with the biotin linker and labelling the second sample with a deuterated form of the biotin linker. Each peptide in the samples is then represented as a pair of peaks in the mass spectrum. Integration of the peaks in the mass spectrum corresponding to each tag indicate the relative expression levels of the peptide linked to the tags.
Selected reaction monitoring (SRM) and multiple reaction monitoring (MRM) provide highly selective methods of tandem mass spectrometry which have the potential to effectively filter out all molecules and contaminants except the desired analyte. This is particularly beneficial if complex samples are analysed which tend to have several isobaric species present within a defined analytical window. Usually, a combination of precursor (parent ion) selection in the first stage of the mass spectrometer (here termed Q1: quadrupole 1, but also equivalent for the respective stages in non-quadrupole mass spectrometers such as ion traps etc.), fragmentation of the parent ion into many fragments of which one or several specific fragments are selected in the following steps of the MS-measurement (usually in quadrupole 3, Q3) and detected at the ion detector. This two-step selection ensures that the desired analyte is measured and any other ion species are reduced in their intensity. Signal-to-noise ratio is much superior to conventional MS/MS experiments which select one mass window in Q1, and then measure all generated fragments in the ion detector. In principle, this MS-based approach can provide absolute structural specificity for the analyte, and in combination with appropriate stable isotope-labelled internal standards (SISs), it can provide absolute quantitation of analyte concentration.
In conventional SRM/MRM type experiments, a stable isotope labelled reference is used to generate an analyte/reference pair which will be used for quantification of analyte against the reference. For the analysis of proteins, such a reference peptide differs from the analyte to be measured only by incorporation of isotopes, to make it distinctly different in mass for the Q1 selection, but otherwise identical in chemical composition, and physico-chemical behaviour. In a typical experiment, the analyte/reference pair are selected, i.e. in Q1 by switching mass selection channels between these two masses. The subsequent fragmentation of these two ions leads to distinct (specific) fragment masses. One or more suitable fragment masses are then chosen where the Q3 filter remains on the position of the selected fragment ions, thus assuring transition of this ion to the mass analyser, and filtering out other ion species.
Recent work in designing improved mass labels for identifying analytes using mass spectrometry has focused on mass labels which are more easily identified in the mass spectrum without other contaminants.
WO 01/68664 discloses a set of two or more mass labels, each label in the set comprising a mass marker moiety attached via a cleavable linker to a mass normalisation moiety, the mass marker moiety being fragmentation resistant. The aggregate mass of each label in the set may be the same or different and the mass of the mass marker moiety of each label in the set may be the same or different. In any group of labels within the set having a mass marker moiety of a common mass each label has an aggregate mass different from all other labels in that group, and in any group of labels within the set having a common aggregate mass each label has a mass marker moiety having a mass different from that of all other mass marker groups in that group, such that all of the mass labels in the set are distinguishable from each other by mass spectrometry. This application further discloses methods of analysis comprising detecting an analyte by identifying by mass spectrometry a mass label or a combination of mass labels unique to the analyte. Tandem mass spectrometry may be used. Specifically, the mass spectrometer employed to detect the mass label may be a triple quadrupole mass analyser comprising a first analyser to select ions of a particular mass or mass range, a second mass analyser to dissociate the selected ions and a third mass analyser to detect resulting ions.
WO 03/025576 discloses a set of two or more mass labels, each label in the set comprising a mass marker moiety attached via at least one amide bond to a mass normalisation moiety. The mass marker moiety comprises an amino acid and the mass normalisation moiety comprises an amino acid. As for WO 01/68664 the aggregate mass of each label in the set may be the same or different and the mass of the mass marker moiety of each label in the set may be the same or different such that all of the mass labels in the set are distinguishable from each other by mass spectrometry. As for WO 01/68664 this application also discloses a method of analysis which may involve tandem mass spectrometry. This application is specifically directed to the analysis of peptides and mass labels with mass normalisation moieties and mass marker moieties comprising at least one amino acid.
WO 2007/012849 discloses a mass label and a reactive mass label having a general chemical formula for labelling and detecting a biological molecule by mass spectroscopy. The mass labels and reactive mass labels of this invention are clearly identified in a mass spectrum and are easily reacted with analytes. As for WO 01/68664 this application also discloses a method of analysis which may involve tandem mass spectrometry.
The development of isobaric mass tags in the late 1990's has revolutionised biomarker discovery. The ability to analyse multiple samples in theoretically unlimited numbers in a single LC-MS/MS workflow increases throughput whilst at the same time reducing analytical variability. Therefore, there remains a need for improved methods of quantitatively detecting and routinely measuring analytes by mass spectrometry in a wide range of samples.